IJPST Volume 4, Nomor 2 , Juni 2017

THE EFFECT OF EXTRACTION CONDITION ON THE POLYPHENOL CONTENT AND ANTIOXIDANT ACTIVITY OF Curcuma zedoaria (Christm.) ROSCOE RHIZOME

Lia Marliani, Wempi Budiana, dan Yonara Anandari Sekolah Tinggi Farmasi Bandung, Bandung, Indonesia

ABSTRAK White turmeric (Curcuma zedoaria (Christm.) Roscoe) is one of Indonesian herbal medicine. The extraction process to get polyphenol compound from natural product was influenced by some factor such as solvent, temperature, time, and method of extraction. The objective of this study was to determine the significant factor of extraction that is solvent, temperature and time of extraction on the polyphenol content and antioxidant activity of white turmeric (Curcuma zedoaria (Christm.) Roscoe) rhizome. Extraction was done by dynamic maceration method with variations (23 factor variable design) of solvent (ethanol 96% and water), temperature (25°C and 70°C), time (6 and 24 hours). The method of analysis of polyphenol content using Folin Ciocalteu reagent, and the antioxidant activity using DPPH free radical reduction method. The experiment design and data analysis using Design-Expert® Software Version 10. The result showed that extraction using ethanol 96% at 70°C for 24 hours was gave high polyphenol content and antioxidant activity. Data analysis was showed that polyphenol content and antioxidant activity was influenced only by solvent of extraction. This study indicated that solvent is significant extraction factor for polyphenol content and antioxidant activity of white turmeric (Curcuma zedoaria (Christm.) Roscoe) rhizome.

Keywords: Antioxidant, Curcuma zedoaria (Christm.) Roscoe, extraction, polyphenol

Pengaruh Kondisi Ekstraksi terhadap Kandungan Polifenol dan Aktivitas Antioksidan Rimpang Curcuma zedoaria (Christm.) Roscoe

ABSTRACT Temu Putih (Curcuma zedoaria (Christm.) Roscoe) adalah salah satu obat herbal Indonesia. Proses ekstraksi untuk mendapatkan senyawa golongan polifenol dipengaruhi oleh beberapa factor seperti pelarut, suhu, waktu, dan metode ekstraksi. Tujuan penelitian ini adalah untuk menentukan faktor ekstraksi (pelarut, suhu, dan waktu ekstraksi) yang signifikan terhadap kandungan polifenol dan aktivitas antioksidan dari rimpang temu putih (Curcuma zedoaria (Christm.) Roscoe) rhizome. Ekstraksi dilakukan dengan metode maserasi dinamik dengan variasi (variasi desain faktorial 23) pelarut (etanol 96% dan air), suhu (25°C and 70°C), waktu (6 dan 24 jam). Penetapan kadar polifenol menggunakan reagen Folin Ciocalteu dan aktivitas antioksidan menggunakan metode reduksi radikal bebas DPPH. Design eksperimen dan analisis data menggunakan Software Design-Expert® Version 10. Hasil penelitian menunjukkan bahwa ekstraksi menggunakan ethanol 96% pada suhu 70°C selama 24 menghasilkan kandungan poliphenol dan aktivitas antioksidan tertinggi. Analisis Data menunjukkan bahwa kandungan Polifenol dan aktivitas antioksidan hanya dipengaruhi oleh pelarut ekstraksi. Penelitian ini menunjukkan bahwa pelarut merupakan faktor ekstraksi yang signifikan terhadap kandungan polifenol dan aktivitas antioksidan rimpang Temu Putih (Curcuma zedoaria (Christm.) Roscoe).

Kata kunci : Antioksidan, Curcuma zedoaria (Christm.) Roscoe, ekstraksi, polifenol

Korespondensi: Lia Marliani 57 [email protected] IJPST Volume 4, Nomor 2 , Juni 2017

Introduction compound was extracted. The purpose of White turmeric (Curcuma zedoaria this study was to determine the significant (Christm.) Roscoe) is one of family Zingiberaceae that usually used as one of extraction factors that influence the Indonesian herbal medicine. Its rhizome polyphenol content and antioxidant activity has antioxidant, anti-inflammatory, anti- of white turmeric (Curcuma zedoaria cancer and anti-microbial activity 1. White (Christm.) Roscoe). turmeric contains bioactive compounds Materials and Methods such as flavonoids, polyphenols, Materials curcuminoid, terpenoids and essential oils. White turmeric (Curcuma zedoaria There are approximately 60 essential oil (Christm.) Roscoe) rhizome were collected components and oleoresin in white turmeric from Purwakarta, West Java, Indonesia, at with curzerenone, germacrone, camphor, Februari 2016. The specimen were curcumenol as the largest component 2. identified at Herbarium Biology The one of antioxidant compound Department, Padjadjaran University, of white turmeric is polyphenols. Bandung, Indonesia. Sample was dried by Polyphenols has been known as potent oven at temperature 50⁰C for 2 days. Dry antioxidant. Polyphenols can scavenge material grinded and was sieved by mesh radical species or inhibit some enzymes or 20 sieve. chelating trace metals which involve in free Extraction radical formation 3. Sample was extracted by dynamic The extraction process aims to maceration method using 23 variable factor obtain maximum active compounds and the (two levels of three factor) includes best quality in activities. The yield of variation of solvent (ethanol 96% and extraction is influenced by the type of water) in 200 mL, temperature (25oC and extraction method, solvent, extraction time, 70oC) and time (6 hours and 24 hours). The and temperature 4,5,6. There is no universal extraction process was done in an orbital extraction method for all phenolic shaker at stirring speed of 30 rpm. Each compounds because their solubility and extract was concentrated using Buchi® physical characteristics varying as well as Rotavapor® R-215 to obtain spissum varying form of phenolic compounds from extract. simple to highly polymerized substances 3. Determination of total phenol content Total phenol content and total flavonoid Total phenolic compounds contents content of the extract are varying depend on were determined by the Folin-Ciocalteau extraction solvent 7. The higher amounts of method 10. The 0.5 ml extract samples were phenolic compound often extracted by mixed with 5 ml Folin Ciocalteu reagent for more polar organic solvent such as 5 minute and Na2CO3(4 ml, 1 M) then methanol and ethanol. The amount of added. The mixture was allowed to stand polyphenols extracted are influenced by for 15 min and the phenols were determined selecting the right solvent 8. According to by colorimetry at 765 nm. The standard was Wissam et al, 2012 9, an increasing 20, 40, 50, 60, 80, and 100 µg/ml gallic temperature of extraction will increases the acid. Total phenol values are expressed in extraction efficiency by increases cell terms of gallic acid equivalent (µg/ml of permeable, solubility and diffusion dry mass), which is a common reference coefficients of the compounds. compound μg −3 푚𝑔 퐺푎푙푙𝑖푐 푎푐𝑖푑 푒푞푢푣푎푙푒푛푡 ( ). 10 The antioxidant activities are 푇표푡푎푙 푝ℎ푒푛표푙 푐표푛푡푒푛푡 ( ) = ml 퐺 μg −6 depending on the level of antioxidant 푆푎푚푝푙푒 푐표푛푐푒푛푡푟푎푡𝑖표푛 (ml) . 10

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Determination of antioxidant activity temperature and time that influence the The free radical scavenging activity total phenols content, and antioxidant of extract was determined using the stable activity. radical DPPH (1,1‐diphenyl‐2‐ The mathematical models for each picrylhydrazyl) 10,11. Different response were evaluated using a multiple concentration of each extract (50-250 regression method. The response function µg/ml for ethanol 96% extract and 500- applied was a linear polynomial equation, 1000 µg/ml for water extract) were added at given by equation (1): equal volume (1:1) of freshly prepared DPPH solution (60 µg/ml) in methanol in each test tube and mixed. The absorbance …..(1) was measured after 30 min incubation at In equation (1), Y is the dependent 516 nm (λmax of DPPH solution). The variable; β0 is the constant term; k number capability to scavenge the DPPH radical of variables; βi represents the coefficients was calculated using the following of linear parameters; βij represents the equation: coefficients of interaction parameters. DPPH scavenged (%) = [(Ac – The significance of the equation As)/Ac]x100 parameters for each response variable was Ac is the absorbance of the control analyzed by F-test. Only the factors with (DPPH solution) and As is the absorbance significance higher than or equal to 5% of the extracts. The antioxidant activity of (p≤0.05) were considered. The model the extract was expressed as IC50. The adequacy was checked accounting for the IC50 value was determined from linear coefficient of determination (R2). The regression analysis with Concentrations of response and result of data analysis by extract as X axis and DPPH scavenged (%) Design-Expert® Software Version 10 was as Y axis. showed by Interaction plot graphic (2D). Data analysis Results and Discussion The results were analyzed by The result of total phenol content Factorial analysis. ANOVA/ Response determination showed that the highest Surface Methodology (RSM) on the content was obtained by extraction using experimental data was performed using ethanol 96% at 24 hours and temperature using Design-Expert® Software Version 10 70ºC (see Table 1). to determine which factors of solvent, Table 1 Yield and Total Phenol Content of Extract Extraction factor variation Total Phenol Temperature Time Yield (%) Content Solvent (⁰C) (hours) (mgGAE/G) Water 25 6 29.40±0.990 20.869 ± 3.648 Water 25 24 28.75±1.060 20.376 ± 2.079 Water 70 6 31.95±2.758 19.945 ± 6.831 Water 70 24 26.08±0.813 23.463 ±2.010 Ethanol 96% 25 6 18.70±0.778 76.879 ± 2.803 Ethanol 96% 25 24 18.05±0.848 84.028 ± 3.834 Ethanol 96% 70 6 23.70±2.616 80.250 ± 5.821 Ethanol 96% 70 24 26.15±5.162 84.150 ± 4.142

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This is accordance with Do et al. maybe the reason why ethanol 96% extract research 7 which its result showed the gave the highest the total phenol content. extract obtained by 100% ethanol has Temperature and extraction time are highest phenolic content than other solvent enhancing the solubility and diffusion and the water extract showed the lowest coefficient of solute 13. The result showed phenolic content. Rhizome may contain that at 70⁰C and 24 hour extraction, both varying phenolic compounds from simple water and ethanol extract gave the highest to polymerized subtances. The other phenolic compound. The chance of compound such as carbohydrates and denaturation and oxidation of phenolics is proteins may also associated with phenolics increased by high temperature and long compound 3. The complex polyphenols are extraction which decrease the yield of more soluble in organic solvent, such as phenolics in the extracts7. The result ethanol than water. The solubility of showed that at 70⁰C and 24 hour extraction, nonphenol compounds like carbohydrate phenolic compound of white turmeric and protein are higher in water than in (Curcuma zedoaria (Christm.) Roscoe) ethanol 12. The water can extract other rhizome may not be denaturated or compound of white turmeric such as oxidized. amylum that also abundant in rhizome. This

Design-Expert® Sof tware Factor Coding: Actual Kadar Fenol (mg/G) One Factor

X1 = A: Pelarut 100

Actual Factors B: Suhu Ekstraksi = 44,4595 C: Waktu Ekstraksi = 15 80

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40 Kadar Fenol (mg/G)

20 Total phenol content Total (mgGAE/G) 0

WATEAir Solvent ETHANOEtanol Figure.1 : The graphic of solventR effect to A:total Pelarut phenol Lcontent (Design-Expert®) content and ethanol 96% gave the highest Although at different temperature total phenol content (figure 1). and time of extraction showed different The result of antioxidant activity phenol content, the data analysis by that determined by free radical DPPH Design-Expert® showed only solvent scavenging activity showed the lowest IC50 variation ((significance higher than 5%, p value also was obtained by extraction using p≤0.05) could affecting the total phenol ethanol 96% at 6 hours and temperature 70ºC (see Table 2).

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Table 2 : IC 50 value of Extract Extraction factor variation Temperature Time IC50 (µg/ml) Solvent (⁰C) (hours) Water 25 6 956.16±20.27 Water 25 24 754.80±244.38 Water 70 6 821.18±107.20 Water 70 24 756.13±77.83 Ethanol 96% 25 6 203.12±9.69 Ethanol 96% 25 24 194.70±0.40 Ethanol 96% 70 6 184.25±6.35 Ethanol 96% 70 24 210.12±5.13

The lowest IC50 value determine the white turmeric (Curcuma zedoaria more potent antioxidant activity. This result (Christm.) Roscoe). Although at different (Table 1 and 2) showed that the high total temperature and time of extraction showed phenol content gave the most active different phenol content, but they are not antioxidant. It is also determined the role of significant. polyphenol as antioxidant compound of

Design-Expert® Sof tware Factor Coding: Actual Original Scale One Factor IC 50 (ppm) 1000 X1 = A: Pelarut

Actual Factors B: Suhu Ekstraksi = 47,5 C: Waktu Ekstraksi = 15 800

600

(µg/ml) 400

IC 50 (ppm) 50 IC IC 200

0

WATEAir Solven ETHANEtanol R OL Figure.2 : The graphic of solvent effectt A: to Pelarut IC50 value (Design-Expert®)

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The data analysis by Design- concentration, extraction time and Expert® determines that only solvent extraction temperature on the variation (significance higher than 5%, recovery of phenolic compounds p≤0.05) could influence the antioxidant and antioxidant capacity of activity (figure 2). The potent antioxidant Orthosiphon stamineus extracts. showed by extract which obtain using International Food Research organic solvent (ethanol 96%). Journal. 2011; 18: 1427-1435. Conclusion 6. Ng L.Y., Ang Y.K., Khoo H.E. and This study showed that solvent is Yim H.S. Influence of different significant extraction factor for polyphenol extraction parameters on content and antioxidant activity of white antioxidant properties of Carica turmeric (Curcuma zedoaria (Christm.) papaya peel and seed. Research Roscoe) rhizome than temperature and time Journal of Phytochemistry. 2012; 6: of extraction. 61-74. 7. Do QD, Angkawijaya AE, Tran- Acknowledgement Nguyen PL, Huynh LH, Soetaredjo FE, Ismadji S, Ju YH. Effect of This research was supported by an Internal extraction solvent on total phenol Grant from Bandung School of Pharmacy, content, total flavonoid content, and Bandung, Indonesia. antioxidant activity of Limnophila aromatic. Journal of Food and Drug References Analysis. 2014; 22: 296-302 1. Lobo R, Prabhu KS, Shirwaikar A, 8. Xu B.J., Chang S.K. A. comparative Shirwaikar A. Curcuma zedoaria study on phenolic profiles and Rosc. (white turmeric): a review of antioxidant activities of legumes as its chemical, pharmacological and affected by extraction solvents. ethnomedicinal properties. Journal Journal of Food Scince. 2007; 72: of Pharmacy and Pharmacology. S159-166. 2009; 61: 13–21. 9. Wissam Z., Bashour Ghada B., 2. Singh P, Singh S, Kapoor IPS, Wassim A., Warid K. Effective Singh G, Isidorov, V, Szczepaniak extraction of polyphenols and L. Chemical composition and proanthocyanidins from antioxidant activities of essential oil pomegranate’s peel. International and oleoresins from Curcuma Journal of Pharmacy and zedoaria rhizomes, part-74. Food Pharmaceutical Sciences. 2012; Bioscience. 2013; 3: 42–48 4(3): 675 - 682. 3. Dai J, Mumper RJ. Plant Phenolics: 10. Ghasemi K, Ghasemi Y, Extraction, Analysis and Their Ebrahimzadeh MA. Antioxidant Antioxidant and Anticancer Activity, Phenol and Flavonoid Properties. Molecules. Contents of 13 Citrus Species Peels 2010,15:7313-7352 and Tissues. Journal and 4. Spigno G, Tramelli L, Faveri Pharmaceutical Sciences. 2009; D.M.D. Effects of extraction time, 22(3): 277-281 temperature and solvent on 11. Seal T. Evaluation of antioxidant concentration and antioxidant activity of some wild edible fruits activity of grape marc phenolics. of Meghalaya state in India. Journal of Food Engineering. 2007; International Journal of Pharmacy 81: 200-208. and Pharmaceutical Sciences. 2011; 5. Chew K.K, Ng S.Y, Thoo Y.Y, 3(4):233-236 Khoo M.Z., Wan Aida W.M, Ho 12. Sultana B, Anwar F, Ashraf M. C.W. Effect of ethanol Effect of Extraction

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Solvent/Technique on the Antioxidant Activity of Selected Medicinal Plant Extracts. Molecules. 2009; 14 : 2167-2180 13. Spigno G., and De Faveri D. M. Antioxidants from grape stalks and marc: influence of extraction procedure on yield, purity and antioxidant power of the extracts. Journal of Food Engineering. 2007; 8:793–801.

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